The present invention pertains generally to methods and apparatus for analytical chemistry, and more particularly to test kits and methods for qualitatively or quantitatively determining one or more analytes present within a matrix such as a food substance or biological fluid.
It is routinely desirable to test for the presence of specific analytes in substances which are intended for human consumption or application to the human body (e.g., foods, beverages, cosmetics, toiletries, topical solutions, contact lens solutions, pharmaceutical preparations, etc.) to confirm that such substances are fresh (i.e., not degraded), pure and free of contamination. Additionally, it is often desirable to test for the presence of specific analytes in samples of biological fluids (e.g., blood, plasma, serum, urine, saliva, bile, lymph, etc.) which have been extracted from the human body.
However, the analytical techniques which have heretofore been utilized to quantitatively or qualitatively test for specific analytes in complex matrices are often problematic, due to the fact that such substances may contain many diverse physical and/or chemical species, some or all of which may interfere with the intended analysis. Thus, it is frequently necessary for the test substance to be subjected to extensive sample preparation steps, in order to isolate and/or concentrate the particular analyte(s) of interest, prior to actually proceeding with analytical determination of the desired analyte(s). Moreover, in instances where the test substance is a solid material (e.g., food) it is often necessary to chop or grind the solid material into particles, and to extract the desired analyte(s) from such particles by adding one or more liquid digestants, solvents or other fluids to form a slurry or suspension, and thereafter performing a xe2x80x9cclean upxe2x80x9d of the slurry or suspension by filtration or centrifugation to separate the analyte containing liquid from the extraneous solid matter.
In instances where multiple analytes are to be determined, it is often necessary to perform several separate, time consuming, analytical procedures (e.g., gas chromatography (GC), high performance liquid chromatography (HPLC) or other analytical chemistry procedures) on aliquots or extracts of the test substance, in order to generate the desired multiple analyte data.
Thus, the traditional methods for determining the presence of, or detecting specific analyte(s) in complex matrices (e.g., substances which contain matter other than the desired analyze(s)) can be quite time consuming, skill intensive and expensive.
It is frequently desirable to detect or quantify, in foods, one or more particular analyte(s) which are indicative of the freshness or quality of the food. In routine quality control testing of foods ,it is common practice to test for the presence of various contaminates, additives, degradation products, and/or chemical markers of microbial infestation (e.g., bacterial endotoxins, mycotoxins, etc . . . ). However, the current methods by which such quality control testing of food is accomplished are typically either: a) complex and skill-intensive analytical chemistry procedures or b) highly subjective and qualitative sensory evaluations (e.g., smell test, taste test, appearance, etc.).
As fatty acids within foods oxidize, relatively unstable lipid hydroperoxides are formed. The presence of these lipid hydroperoxides typically do not affect the smell or flavor of the food in any discernible way. These lipid hydroperoxides then further decompose to form relatively stable lipid aldehydes (e.g., malonaldehyde). The accumulation of lipid aldehydes within the food can give rise to off-odors and off-flavor of the food. Thus, it is difficult or impossible to detect the presence of abnormally high lipid hydroperoxide levels in foods by smell or taste testing, despite the fact that such elevated lipid hydroperoxide levels may indicate that the fats of the food have begun to undergo oxidative degradation and are becoming rancid. Moreover, inadvertent consumption of. these undetected lipid hydroperoxides may adversely affect the health of human beings due to the fact that such hydroperoxides are believed to play a significant role in the pathogenesis of atheroschlortic vascular disease and/or other health problems.
Various analytical techniques have previously been available to detect the presence of the lipid hydroperoxides and/or lipid aldehydes in foods, many of which involve the separate steps of a) extraction, b) clean-up, c) derivitization, d) analysis and e) detection. These previously utilized analytical techniques for detecting lipid hydroperoxides and lipid aldehydes in foods are typically expensive, time consuming, and require considerable expertise and training.
In particular, one frequently used analytical procedure for lipid aldehydes, known as the thiobarbituric acid (TBA) assay, requires that the lipid aldehydes be extracted and isolated in an analytical solution and subsequently reacted with thiobarbituric acid to give a red fluorescent adduct, which exhibits maximum UV absorbance at 532 nm. The initial extraction and isolation of the lipid aldehydes frequently requires laborious sample preparation steps. Moreover, the TBA assay is not specific for malonaldehyde (the primary lipid aldehyde in rancid fats), but rather may react with other aldehydes or other chemical species which are not indicative of rancidity. Thus, the reliability and meaningfullness of the TBA assay for assessing rancidity in foods is controversial.
Other, more complicated analytical methods have been utilized to detect lipid hydroperoxides and/or rancidity-indicating aldehydes in foods, including procedures based on electron spin resonance, high-performance liquid chromatography, and liquid chromatography-chemiluminescence techniques. However, these other analytical methodologies for assessing rancidity of fats can be extremely expensive, time consuming, and labor-intensive.
Examples of previously-known analytical techniques or other evaluations for determining lipid aldehy des in foods or other complex matrices include those described in the following publications: Nollet, L.ML.(ed.), Handbook of Food Analysis, Marcel Decker, Inc. (1996); Warner, K., Sensory Evaluations Based on Odor and Flavor: Methods to Assess Quality and Stability of Oils and Fat Containing Foods, Pgs. 49-75, AOCS Champaign Il. (1995); Evans, C. D., Analysis of Headspace Volatiles by Gas Chromatography, Proceedings of AOCS October Meeting (Pg. 15-18) (1967); Dugan, L., Kreis Test for Cxe2x95x90O Groups With Phloroglucinol, Journal of the American Oil Chemists Society 32, Pg. 605 (1955).
Examples of previously-known methods for determining lipid peroxides in foods or other complex matrices include those described in the following publications: Nollet, L. M. L. (ed), Handbook of Food Analysis, Marcel Decker, Inc. (1996); Methods to Determine Lipid Peroxides by Titration Method, Journal of the American Oil Chemists Society, Vol. 26, Pg., 345 (1949); Gray, J. I., Conjugated Diene Measurements at 230-375 nm, Journal of the American Oil Chemists Society, Vol. 45, Pg. 632 (1978), Halliwell B, Gutteridge J M C. Free radicals in biology and medicine, 2nd ed. Oxford,d UK: University Press, 1989:543pp; Gutteridge J M C, Halliwell B. The measurement and mechanisms of lipid peroxidation in biological systems, Trends Biochem Sci 1990;15:129-35; Gutteridge J M C. Lipid peroxidation: some problems and concepts, in ed. (Oxygen radicals and tissue injury). Halliwell B., Bethesda, M D:FASEB, 1977:9-19; Gutteridge J M C, Kerry P J. Detection by fluorescence of peroxides and carbonyls in samples of aracyidonic acid. Br J Pharmacol 1982;76:459-61; Gutteridge J M C. Iron promoters of the Fenton reaction and lipid peroxidation can be released from haemoglobin by Peroxides. FEBS Lett 1986;20:291-5. ; Gutteridge J M C, Beard A P C, Quinlan G J. Superoxide-dependant lipid peroxidation: problems with the use of catalase as a specific probe for Fenton-driven hydroxyl radicals. Biochem Biophys Res Commun 1983;117:901-7. ; Halliwell B, Gutteridge J M C. Lipid peroxidation, oxygen radicals, cell damage and antioxidant therapy. Lancet 1974;1:1396-8; Halliwell B, Gutteridge J M C greater than  The definition and measurement of antioxidants in biological systems. Free Radic Bio Med 1995;18:125-6; Gutteridge J M C. The antioxidant activity of haptoglobin towards haemoglobin stimulated lipid peroxidation. Biochim Biophys Acta. U.S. Pat. No. 5,320,725, entitled xe2x80x9cElectrode and method for the detection of hydrogen peroxide,xe2x80x9d (Gregg et al.), Assignee: E. Heller and Company, Austin, Texas; U.S. Pat. No. 4,851,353, entitled xe2x80x9cMethod and test composition for determination of lipid peroxide,xe2x80x9d (A. Miike, et al.), Assignee Kyowa Hakko Kogyo Co., Ltd., Tokyo, Japan; U.S. Pat. No. 4,900,680, entitled xe2x80x9cMethod and apparatus for measuring lipid peroxide,xe2x80x9d (T. Miyazawa, et al.), Assignee: Tobuku Electronic Industrial Co., Ltd., Sendai, Japan; U.S. Pat. No. 5,061,633, entitled xe2x80x9cMethod for analyzing lipid peroxides using aromatic phosphinesxe2x80x9d, (H. Meguro, et al.), Assignee: Tosoh Corporation, Japan; U.S. Pat. No. 4,367,285, entitled xe2x80x9cAssaying lipid peroxide in lipid composition,xe2x80x9d U.S. Pat. No. 4,367,285, entitled xe2x80x9cAssaying lipid peroxide in lipid composition,xe2x80x9d (T. Yamaguchi, et al.), Assignee: Toyo Jozo Company, Ltd., Tokyo, Japan; U.S. Pat. No. 4,657,856, entitled xe2x80x9cGlutathione peroxidase, process for production thereof, method and composition for the quantitative determination of lipid peroxide,xe2x80x9d (O. Terada, et al.), Assignee: Kyowa Hakko Xogyo Co., Ltd., Tokyo, Japan.
The analysis of lipid peroxides and/or lipid aldehydes in foods or other matrices is not limited to applications wherein it is desired to determine whether the food or other matrix has undergone oxidative degradation. In fact, it is often desirable to test for lipid peroxides and/or lipid aldehydes as a means of determining the resistance to oxidation or xe2x80x9cantioxidant statusxe2x80x9d of a particular food product or other formulation. Such testing for antioxidant status provides a means for determining whether a food or other type of product is likely to undergo oxidative degradation under the production, shipping, storage and use conditions to which the food or other product will be exposed. In order to mimic extreme oxidative conditions, such testing for antioxidant status is often performed in conjunction with an oxidative challenge, such as the purposeful addition of an oxidation promoting chemical to the test formulation, or by exposing the test material to high-intensity light or heat.
In this regard, antioxidants are often added to food products, cosmetics or other formulations to prevent oxidative degradation or deterioration during production, storage and/or cooking. It is critical, however, that such antioxidant additives be present at sufficient concentrations to prevent potentially toxic lipid peroxides and/or aldehydes from forming under the intended production, storage and/or cooking conditions. Thus, in the development of food and/or other product formulations it is often necessary to test various types, combinations and/or concentrations of antioxidant additives in order to determine which formulation(s) are best suited for the intended production, storage and/or cooking conditions. Moreover, it is often desirable to perform analyses of lipid peroxide and/or lipid aldehyde concentrations in previously-prepared food and/or product formulations as a means of identifying and testing new synthetic and/or natural antioxidants which may be usable to prevent oxidative degradation of such products.
To fully understand the propensity for and state of oxidative degradation of a material (e.g., a food), it is desirable to assay the material for lipid peroxide concentration, lipid aldehyde concentration, and resistance to oxidation, at least two (2) temperatures, at 2 or more time points over 0 to 48 hours. The evaluation temperatures may typically include 56xc2x0 C. and 37xc2x0 C., since these temperatures approximate the extremes of usual shelf life conditions. Higher temperatures cause changes in the dynamics of lipid peroxide and lipid aldehyde formation. The time to reach the end points of sudden increases in lipid peroxide and/or lipid aldehyde concentrations is predicative of resistance to oxidation. Also, lipid peroxides are more stable in some matrices than others, so the profile of their values over time, and the relative increase or decrease of their breakdown products, provides complete information about the status of oxidative degradation of the matrix.
When used in foods, the quantity of some antioxidant additives may be subject to governmental regulation, especially in formulations wherein synthetic antioxidant additives are being utilized. Thus, in such situations, it is typically desirable to perform lipid peroxide and/or lipid aldehyde analyses as means of determining the minimum amount(s) of particular antioxidant additives which must be added to a particular formulation to provide the desired antioxidant affect and/or to identify non-regulated natural alternatives to governmental regulated synthetic additive. Thus, the detection and/or analysis of lipid peroxides and lipid aldehydes in foods and other formulations is often carried out for various product/formulation development or research purposes, as well as for quality control testing of the freshness and wholesomeness of the food or other product.
Because the previously-known analytical methods for determining lipid aldehyde and/or lipid peroxide concentrations in foods have involved relatively complex chemical analytical procedures which may be too complex or too skill-intensive for untrained personnel, there exists a need in the art for the development of simple test kits capable of rapidly and reproducible determining the presence and/or concentrations of lipid peroxides and lipid aldehydes in foods and other complex matrices, so that relatively untrained -personnel may perform such determinations in a reliable, cost effective manner.
Many types of chemical contaminants, such as pesticides, herbicides, excessive concentrations of food additives, etc., may be present in foods. It is highly desirable to detect the presence of such chemical contaminants prior to sale or consumption of the affected foods. Unfortunately, the analytical methodologies which have heretofore been utilized for determining the presence of such chemical contaminants in foods have typically required laborious, skill-intensive analytical chemical procedures which are too complex or too skill-intensive to be performed by untrained personnel.
Examples of the types of analytical chemical procedures which have heretofore been utilized to quantitatively or qualitatively determine the presence of chemical contaminants (e.g., herbicides, pesticides, additives) in food include those described in the following publications: Monier, W. G., Williams Determination of Sulfite, Analyst, Vol. 52, Pg. 415, (1927); Rothenfusser, S., Lebensm Untero Forsch, Vol. 58, Pg. 98 (1929); Nollet, L. M. L.(ed.), Handbook of Food Analysis, Marcel Decker, Inc., Pg. 507, (1996); Tekel, J. et al., HPLC Analysis of Herbicides, Journal of Chromatography, Vol. 643, Pg. 291, (1993).
Modern veterinary practice utilizes various drugs and pharmaceutical agents which, when administered to cattle, dairy cows, chickens and other farm animals, will maximize and improve the rate of growth and/or productivity of such animals. For example, antibiotics, corticosteroids and certain beta-adrenergic agonists are sometimes administered to meat-producing animals (e.g., cattle, hogs, chickens, lambs) to accelerate weight gain. Similarly, antibiotics are sometimes administered to farm animals as prophylaxis against or treatment for infectious disease (e.g., mastitis in dairy cows). It is typically necessary to cease administration of these pharmaceutical agents a specified time period prior to slaughtering of the animal or obtainment of food products (milk, eggs) therefrom, to ensure that the meat or other animal products will not contain excessive or potentially toxic levels of these pharmaceutical agents. Thus, it is desirable to routinely test the meats and other food products obtained from drug-treated animals to confirm that such meats and/or food products are not contaminated with excessive levels of these pharmaceutical agents.
The analytical procedures which have heretofore been utilized to determine the concentrations of drugs such as antibiotics, corticosteroids, and/or beta-adrenergic agonists in meats or animal products (e.g., milk, eggs) have been relatively complex, time-consuming and skill-intensive procedures. Examples of previously known analytical procedures for determining the concentrations of antibiotics, corticosteroids, and/or beta-adrenergic agonists in meats or other animal products include those described in the following publications: Cole, R.J.(ed.), Modern Methods in Analysis and Structural Elucidation, Pg. 239, 265, 293, Academic Press (1986); Boison, J. O., Analysis Myrotoxins, Journal of Chromatography, Vol. 629, Pg. 171, (1992); Adams, A. et al., Proc. 2nd International Symposium on Hormone and Veterinary Drug Residue Analysis, Pg. 50, (1994); and, Tomlin, Ct.(ed), British Crop Protection Council; Farniham, Surrey, U.K. (1994).
Some microbes, including certain viruses, bacteria and fungi are known to secrete toxins, enzymes or other chemical markers which may be directly toxic to humans if consumed and/or are clearly indicative of the presence of such microbial contamination in a particular foods. Examples of such chemical markers of microbial contamination include clostridium botulinum toxins, toxins secreted by fusarium T2 and zearalenone fungi which affect corn and other grains, and endotoxins or metabolites given off by certain pathogenic bacteria (e.g., salmonella, lysteria, E. Coli, etc.).
Standard microbiological culture techniques can sometimes be utilized to identify the presence of microbial contaminants in foods, but such microbiological culture techniques typically must be performed by highly trained individuals, and often require a relatively long incubation time.
Similarly, analytical chemical methods can be used for determining or quantifying the presence of the chemical markers (e.g., endotoxins, toxins, metabolites, etc.) of certain pathogenic microbes, but such chemical analytical procedures are also relatively complex, time consuming, and require a substantial amount of technical skill and training.
In view of the foregoing problems, limitations and needs associated with detection and/or quantification of specific analytes (e.g., detection of degradation products, antioxidant status, drug residues, chemical contaminants or markers of microbial contamination, in foods or other matrices) in complex matrices there exists a need in the art for the development of simplified, cost-effective, reliable and reproducible methods and apparatus for performing such detectings and/or quantifications in complex matrices (e.g., foods, biological fluids, etc.).
The present invention provides test kits for qualitatively or quantitatively determining one or more analytes in matrices such as foods, other solid materials or some biological fluids.
In accordance with one embodiment of the invention, there is provided a simple test kit for determining the presence of a single analyte, such test kit comprising; a) a sample receiving vessel, b) a membrane and c) a reagent-containing well. The test sample is initially prepared (e.g., chopped or ground if a solid) and is deposited in the sample-receiving vessel along with any desired diluent, digestion solution (e.g., enzymes), chelators, or chemical modifiers (e.g., antioxidants). The prepared sample is then permitted to drain from the sample-receiving vessel, through the membrane. The type of membrane utilized in each embodiment will be selected based on the type and quantity of matter which is desired to be excluded from the prepared sample matter prior to analysis. In many applications, this initial membrane will be formed of microporous film having pores which are sized to present large particles of solid matter, proteins and other unwanted matter from passing therethrough, but which will allow a filtrate containing the desired analyte to drain into the reagent-containing well. When drained into reagent-containing well, the analyte contained within the filtrate will react with the reagent in a manner which will permit the presence or quantity of analyte to be determined. In many instances, the analyte-reagent reaction will be a color forming reaction such that a visual determination may be made as to whether, or to what degree the desired analyte is present. In other instances, it may be desirable to utilize an analytical instrument to determine the quantity of analyte present in the analyte present in the analyte-reagent solution.
In accordance with other embodiments of the invention, the simple test kit of the above-described character may be adapted for determination of two or more analytes by the addition of one or more additional membranes in series with the first membrane. Each of these additional membranes is operative to capture and hold at least one analyte, while allowing a filtrate containing one or more other analyte(s) to pass therethrough. Each of these additional membranes may subsequently be exposed to a wash or flush solution such that one or more eluants containing each of the additional analytes may be obtained. Each such eluant may subsequently be combined with a reagent to provide an eluant-reagent admixture from which at least one analyte may be determined. In this manner, the present invention is adaptable for the qualitative or quantitative determination of two or more analytes from a single sample.
Further in accordance with invention, there is provided a method and apparatus for determining one or more analytes which are present in a matrix at low (e.g., sub-detectable) levels. This embodiment of the invention may be in the form of test kit comprising a) a sample receiving vessel, b) an analyte-capturing membrane, and c) a reagent-containing well. A sample containing the analyte is passed through the analyte-capturing membrane such that the desired analyte will be captured by the membrane. Thereafter, a known volume of flush solution is utilized to elute the analyte from the membrane and into the reagent-containing well. In this manner, the analyte will become concentrated in a smaller volume than that of the original matrix, thereby providing an eluant-reagent admixture from which the analyte may be qualitatively or quantitatively determined.
Further objects and advantages of the invention will become apparent to those skilled in the art upon reading and understanding of the following detailed description and consideration of the accompanying drawings.